The main objective of this study is to characterize the mechanisms underlying the regulation of c-myc stability. C-myc is a key regulator of cellular processes, including proliferation, apoptosis, differentiation, and transformation. Its expression level is deregulated in a number of human cancers, including B cell lymphomas. The effects of various types of stress (UV, osmotic shock, and cytokines) will be assessed in normal and transformed cells, and the kinases contributing to altered stability will be examined. This will be assessed via half-life determination using pulse-chase upon c-myc cotransfection with stress responsive kinases (e.g. ASK, MEKK1, MEK, JNK and IKK) and non-stress responsive kinases (e.g. GSK3). In vivo and in vitro ubiquitination assays will be used to assess the level of c-myc degradation elicited by stress and/or kinases. The mechanisms by which the aforementioned kinases influence c-myc stability will be analyzed via the creation of c-myc mutants lacking putative binding sites and phosphoacceptor sites for specific kinases. In vitro and in vivo ubiquitination assays, binding, and phosphorylation assays will be performed. To assess the biological ramifications of altered c-myc stability, the effects on proliferation, apoptosis, replicative senescence, and transformation in both normal and transformed cell settings will be addressed. Standard assays will be used to examine each of these parameters. The proposed studies will allow the identification of major regulatory components influencing the ability of c-myc to mediate its varied functions in the cell, and will lead to the identification of novel therapeutic targets.